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WORLD INTELLECTUAL PROPERTY ORGANIZATION
International Bureau

(51) International Patent Classification 3
C12N 15/00, 1/20, 1/00

(11) International Publication Number: WO 84/ 02919

(43) International Publication Date: 2 August 1984 (02.08.84)

(21) International Application Number: PCT/US84/00049

(22) International Filing Date: 16 January 1984 (16.01.84)

(31) Priority Application Number: 458,411

(32) Priority Date: 17 January 1983 (17.01.83)

(33) Priority Country:

(71) Applicant: MONSANTO COMPANY fUS/US]; 800
North Lindbergh Boulevard, St. Louis, MO 63167
(US).

(72) Inventors: FRALEY, Robert, Thomas ; 828 Lisakay
Drive, Glendale, MO 63167 (US). ROGERS, Stephen,
Gary ; 312 Sylvester Avenue, Webster Groves, MO
63119 (US).

(74) Agent: BRUSOK, Fabian, X.; 800 North Lindbergh
Boulevard, St Louis, MO 63167 (US).

(81) Designated States: AT (European patent), BE (Euro-
pean patent), CH (European patent), DE (European
patent), FR (European patent), GB (European pa-
tent), JP, LU (European patent), NL (European pa-
tent), SE (European patent).

Published

With international search report.

(54) Title: PLASMIDS FOR TRANSFORMING PLANT CELLS

(57) Abstract

Several plasmids which are useful for genetically
transforming plant ceils. A first plasmid, such as
pMON120, contains a T-DNA border, one or more
marker genes, a unique cleavage site, and a region of Ti
plasmid homology. A gene which is expressed in plant
cells may be inserted into this plasmid to obtain a deriva-
tive plasmid, such as pMON128 which expresses neomy-
cin phosphotransferase in plant cells. The derivative
plasmid is inserted into a suitable microorganism, such
as A. tumefaciens which contains a Ti plasmid. The in-
serted plasmids recombine with the Ti plasmids to form
co-integrate plasmids. Only a single crossover event is re-
quired to create the desired co-integrate plasmid. A.
tumefaciens cells with co-integrate plasmids are selected
and co-cultured with plant cells. The co-integrate Ti plas-
mids enter the plant cells and insert a segment of T-DNA
which does not contain tumorigenic genes into the plant
genome. The transformed plant cell(s) may be regenerat-
ed into a morphologically normal plant which will pass
the inserted gene(s) to its descendants.

ISOLATE DMA SEOUENCE WITH MARKER GENE FOR
SELECTION OF A. TUM. CONTAINING COINTEORATE
PL ASM 1 03

(pMON 109)

ISOLATE DNA SEQUENCE WITH
T-ONA80ROER FROM TI PLAS-

mio ( optional: plant
s cor able marker)

(pMON 41)

ISOLATE DNA SEQUENCE WITH
REGION HOMOLOGOUS TO PART
OF T-ONA OFTI PLASMIO

(pMON

L1C ATE 3 SEQUENCES IN PROPER ORIENTATION | .,

(qMON 120)

INSERT CHIMERIC GENE INTO PLASMIO

INSERT PLASMIO INTO fi.CQUi;
MATE E. COLI!WITH A.TUMEFACIENS

INSERT PLASMIO INTO A.jyMEFACjENS j
CONTAI NING CDISARMEO) TI PLASMID |

S FLEET A. TUMEFACIENS CELLS WITH
CO-INTEGRATED TI PLASMIDS HAVING
CHIMERIC GENES

CO- CULTIVATE A | TUMEFACIENS WITH
PLANT CELLS! ALLOW TRANSFER OF
CO-INTEGRATE PL ASMIOS INTO PLANT
CELLS

CULTIVATE PLANT CELL ^DEN^FY PLANT
CELLS HAVI N GtEXPRE SSION OFJCH I MERIC
GENE OR SCO R ABLE MARKER

REGENERATE PLANT CELLS INTO PLAMTS |

FOR THE PURPOSES OF INFORMATION ONLY

Codes used to identify States party to the PCT on the front pages of pamphlets publish]

ing international ap-

plications under the PCT.

Austria

Republic of Corea

Australia

Liechtenstein

Belgium

Sri t jrifa

Bulgaria

Luxembourg

Brazil

Monaco

Central African Republic

Madagascar

Congo

Mauritania

Switzerland

Malawi

Cameroon

Netherlands

Germany, Federal Republic of

Norway

Denmark

Romania

Finland

Sudan

France

Sweden

Gabon

Senegal

. United Kingdom

Soviet Union

Hungary

Chad

Japan

Togo

Democratic People's Republic of Korea

United States of America

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PLASMIDS FOR TRANSFORMING PL ANT CELLS
Technical Field

This invention is in the fields of genetic
5 engineering, plant biology, and bacteriology.

BACKGROUND ART

In the past decade, the science of genetic
engineering has developed rapidly. A variety of

10 processes are known for inserting a heterologous gene
into bacteria, whereby the bacteria become capable of
efficient expression of the inserted genes. Such
processes normally involve the use of plasmids which
may be cleaved at one or more selected cleavage sites

15 by restriction endonucleases . Typically, a gene of

interest is obtained by cleaving one piece of ON A, and
the resulting DNA fragment is mixed with a fragment
obtained by cleaving a vector such as a plasmid. The
different strands of DNA are then connected

20 ("ligated") to each other to form a reconstituted
plasmid. See, for example, U.S. Patents 4,237,224
(Cohen and Boyer, 1980); 4,264,731 (Shine, 1981);
4,273,875 (Manis, 1981); 4,322,499 (Baxter et al,
1982), and 4,336,336 (Silhavy et al, 1982).

25 A variety of other reference works are

available. Some of these works describe the natural
process whereby DNA is transcribed into mRNA and mRNA
is translated into protein, see, e.g., L. Stryer,
Biochemistry , 2nd edition, p 559 et seq. (W. H.

30 Freeman and Co., 1981); A. L. Lehninger, Biochemistry,
2nd edition, p. 853 et seq. (Worth Publ., 1975).
Other works describe methods and products of genetic
manipulation; see, e.g., T. Maniatis et al,
Molecular Cloning, A Laboratory Manual (Cold Spring

35 Harbor Labs, 1982); J.K. Setlow and A. Hollaender,
Genetic Engineering, Principles and Meth ds (Plenum

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Press, 1979). Hereafter, all references will be cited
in abbreviated form; a list of complete citations is
included after the Examples.

Most of the genetic engineering work
performed to date involves the insertion of genes into
various types of cells, primarily bacteria such as
E. coli , various other microorganisms such as yeast,
10 and mammalian cells. However, many of the techniques
and substances used for genetic engineering of animal
cells and microorganisms are not directly applicable
to genetic engineering involving plants.

15 As used herein, the term "plant" refers to a

multicellular differentiated organism that is capable
of photosynthesis, such as angiosperms and
multicellular algae. This does not include
microorganisms , such as bacteria, yeast, and fungi*

20 The term "plant cell" includes any cell derived from a
plant; this includes undifferentiated tissue such as
callus or crown gall tumor, as well as plant seeds,
propagules, pollen, or plant embryos.

Ti and Ri Plasmids

The tumor-inducing (Ti) plasmid of
Agrobacterium tumefaciens has been proposed for use as
a natural vector for introducing foreign genetic

30 information into plant cells (Hernalsteen et al 1980;
Rorsch and Schilperoort, 1978 ) . Certain types of A.
tumefaciens are capable of infecting a wide variety of
plant cells, causing crown gall disease. The process
of infection is not fully understood. At least part

35 of the Ti plasmid nters the plant cell. Various
m tabolic alterations ccur, and part of the Ti

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plasmid is inserted into the genome of the plant
(presumably into the chromosomes). The part of the Ti
plasmid that enters the plant genome is designated as
"transferred DNA" (T-DNA). T-DNA is stably maintained
5 in the plant DNA (Chilton et al, 1977; Yadev et al,
1980; Willmitzer et al, 1980; Otten et al, 1981).

Research by several laboratories has led to
the characterization of several structural (i.e.,

10 protein coding) genes located in T-DNA (Garfinkel et
al 1981; Leemans et al 1982), as well as other DNA
sequences which appear to serve various other
functions. For example, two sequences referred to as
the "left border" and the "right border" appear to

15 delineate T-DNA and may be involved in the process
whereby T-DNA is transferred into plant chromosomes
(Zambryski et al 1982).

A different species of Agrobacterium , A.

20 rhizogenes , carries a "root- inducing" (Ri) plasmid
which is similar to the Ti plasmid. Infection of a
plant cell by A. rhizogenes causes hairy root disease.
Like the Ti plasmid, a segment of DNA called "T-DNA"
(also referred to by some researchers as "R-DNA") is

25 transferred into the plant genome of an infected cell.

Various other bacteria are also reported to
be capable of causing genetic transformation of plant
cells, including A, rubi and certain bacteria of the ,
30 genus Rhizobia which have been treated with a

mutagenic agent. Hooykaas et al, at page 156 of
Setlow and Holaender, 1979.

As used herein, the term "Ti plasmid"
includes any plasmid (1) which is contained in a
microorganism, other than a virus, which is capable of

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causing genetic transformation of one or more types of
plants or plant cells, and (2) which contains a
segment of DNA which is inserted into a plant genome .
This includes Ri plasmids.
5 -

As used herein, the term "T-DNA" refers to a
segment of DNA in or from a Ti plasmid (1) which has
been inserted into the genome of one or more types of
plant cells, or (2) which is contained in a segment of

10 DNA that is located between two sequences of bases
which are capable of serving as T-DNA borders. As
used herein, the terms "T-DNA border" and "border" are
determined and applied empirically; these terms shall
refer to a sequence of bases which appears at or near

15 the end of a segment of DNA which is transferred from
a Ti plasmid. into a plant genome.
*-

Despite the existing knowledge of T-DNA and
Ti plasmids, no one prior to this invention has been
20 able to utilize these vectors for the introduction of
foreign genes which are expressed in genetically
modified plants, A variety of obstacles to such use
have been encountered in genetic engineering efforts .
Such obstacles include:

1) the large size (approximately 200,000
base pairs) and resulting complexity of Ti plasmids
preclude the use of standard recombinant DNA
techniques to genetically modify and/or insert foreign
30 genes into specific sites in the T-DNA. For example,
there are no known unique restriction endonuclease
cleavage sites in a Ti plasmid (Leemans et al, 1982).

2) the T-DNA, which is inserted into and
expressed in plant cells, contains genes which are
involved in the production of high levels of

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phytohormones in the transformed plant cells (Leemans
et al 1982 ) • The high levels of phytohormones
interfere with the normal metabolic and regenerative
process of the cells > and prevent the formation of
5 pheno typically normal plants from the cells (Braun and
Wood, 1976; Yang et al, 1980)- Exceptions to this are
rare cases where the T-DNA has undergone extensive
spontaneous deletions in planta to eliminate those
genes involved in phytohormone production. Under

10 these conditions, normal plants are reported to be
obtainable at low frequency (Otten et al, 1981).
However, the T-DNA genes involved in phytohormone
production could not be deleted prior to this
invention, since they were very important in the

15 identification and/or selection of transformed plant
cells (Marton et al, 1979).

As described above, simple recombinant DNA
techniques for introducing foreign genes into plasmids

20 are not applicable to the large Ti plasmid. As a

result, several indirect methods have been developed
and are discussed below. The first reported use of
the Ti plasmid as a vector was in model experiments in
which bacterial transposons were inserted into T-DNA

25 and subsequently introduced into plant cells. The
bacterial transposons were reported to be stably
maintained in the plant genome (Hernalsteens et al,
1980; Garfinkel et al 1981). However, in these cases
the transformed tumor tissues were found to be

30 incapable of regeneration into normal plants, and

there was no reported evidence for the expression of
bacterial genes in the plant cells. In addition,
because the insertion of bacterial transposons is
believed to be essentially random, a great deal of

35 effort was required to identify and localize the
position of the inserted DNA in these examples.

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Therefore, this approach is not likely to be useful to
introduce desired genes in a predictable manner into
plants.

5 Other researchers have reported the use of

intermediate vectors which replicate in both E.coli
and A . tumef aci ens ( Matzke and Chilton, 1981; Leemans
et al 1981; Garfinkel et al, 1981). The intermediate
vectors contain relatively small sub fragments of the

10 Ti plasmid which can be manipulated using standard

recombinant DNA techniques*. The sub fragments can be
modified by the deletion of specific sequences or by
the insertion of foreign genes at specific sites* The
intermediate vectors containing the modified T DNA

15 sub fragment are then introduced into A^ tumef aciens by
transformation or conjugation. Double recombination
between the modified T-DNA fragment on the
intermediate vector and its wild-type counterpart on
the Ti plasmid results in the replacement of the

20 wild-type copy with the modified fragment. Cells
' which contain the recombined Ti plasmids can be
selected using appropriate antibiotics.

Various foreign DNA's have been inserted at
25 specific sites in the T-DNA by this method and they

have been reported to be stably transferred into plant
' cells (Matzke and Chilton, 1981, Leemans et al 1981,
1982). However, such foreign genes have not been
reported to be capable of expression in plant cells ,
30. and the transformed plant cells remain incapable of
regeneration into normal plants. Furthermore , in the
procedure described above, it is preferred for a
double crossover event to occur, in order to
substitute the modified DNA fragment for the wild- type
35 copy. A single crossover results in the formation of a
co-integrate plasmid which contains two copies of the

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T-DNA subfragments. This duplication is undesirable
in these methods since homologous recombination, which
can occur in A. tumefaciens cells or in plant cells,
can result in the loss of the inserted foreign
5 gene(s).

A major disadvantage of the above methods is
that the frequency of double recombination is quite
low, about 10" 4 to 10~ 9 (Leemans et al, 1981) and it
10 requires extensive effort to identify and isolate the
rare double-crossover recombinants. As a result, the
number and types of experiments which can be performed
using existing methods for genetically engineering the
Ti plasmid is severely limited*

Other Means for Inserting DNA into Plant Cells

A variety of other methods have been
reported for inserting DNA into plant cells. One such
20 method involves the use of lipid vesicles, also called
liposomes, to encapsulate one or more DNA molecules.
- The liposomes and their DNA contents may be taken up
by plant cells; see, e.g., Lurquin, 1981. If the
inserted DNA can be incorporated into the plant
25 genome, replicated, and inherited, the plant cells
will be transformed. . " ■

Other alternate techniques involve
contacting plant cells with DNA which is coraplexed

30 with either (a) polycationic substances, such as

poly-L-orni thine (Davey et al, 1980), or (b) calcium
phosphate {Krens et al, 1982). Using these
techniques, all or part of a Ti plasmid has been
reportedly inserted into plant cells, causing

35 tumorigenic alteration of the plant cells.

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Another method has been developed involving
the fusion of bacteria, which contain desired
plasmids, with plant cells. Such methods involve
5 converting the bacteria into spheroplasts and

converting the plant cells into protoplasts. Both of
these methods remove the cell wall barrier from the
bacterial and plant cells, using enzymic digestion.
The two cell types can then be used together by
10 exposure to chemical agents, such as polyethylene
glycol. See Hasezawa et al, 1981.

However, all of the foregoing techniques
suffer from one or more of the following problems:

1. transformation efficiencies reported to
date have been very low;

2. only small DNA molecules can be inserted
20 into plant cells;

3. only small numbers of DNA molecules can
be inserted into plant cells; and/or,

25 4. a gene which is inserted into a plant

cell will not be stably maintained by the plant cell^
unless it is incorporated into the genome of the plant
cell / i.e., unless the gene is inserted into a
chromosome or plasmid that replicates in the plant

30 cell.

For these and possibly other reasons, no one
has yet reported expression of a gene inserted into a
plant cell by any of the foregoing techniques, except
35 for the tumorigenic transformations noted above.

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Prior to this invention, no satisfactory
method existed for the creation and identification of
genetically transformed plant cells which could be
5 , routinely regenerated into morphologically normal
"plants.

SUMMARY OF THE INVENTION

This invention relates to several plasmids
10 which are useful for creating transformed plant cells
which are capable of subsequent regeneration into
differentiated, morphologically-normal plants.. This
invention also relates to microorganisms containing
such plasmids, and to methods for creating such
15 plasmids and microorganisms.

This invention involves a first plasmid,
such as pMON120, which has certain desired
characteristics described below. A gene which is

20 capable of being expressed in plant cells may be
inserted into this plasmid to obtain a derivative
plasmid, such as pMON128. For example, plasmid
pMON128 contains a chimeric gene which expresses
neomycin phosphotransferase IT (NPT II), an enzyme

25 which inactivates certain antibiotics . The chimeric
gene is capable of expression in plant cells.

The derivative plasmid is inserted into a
suitable microorganism, such as Agrobacterium
tumefaciens cells which contain Ti plasmids. In the
30 A. tumefaciens cells, some of the inserted plasmids
recombine with Ti plasmids to form a co-integrate
plasmid; this is due to a region of homology between
the two plasmids. Only a single crossover event is
required to create the desired co-integrate plasmid.

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Because of the characteristics of the
inserted plasmid of this invention/ the resulting
co-integrate Ti plasmid contains the chimeric gene
5 and/or any other inserted gene within the T-DNA region
of the co-integrate plasmid. The inserted gene(s) are
surrounded by at least two T-DNA borders, at least one
of which was inserted into the Ti plasmid by the
crossover event. By means of appropriate antibiotics,
10 A. tumefaciens cells which do not have co-integrate Ti
plasmids with inserted genes are killed.

A. tumefaciens cells with co-integrate
plasmids are co-cultured with plant cells, such as

15 protoplasts, protoplast-derived cells, plant cuttings,
or intact plants, under conditions which allow the
.co-integrate Ti plasmids, or portions thereof, to"
enter the plant cells. Once inside the plant cells, a
portion of the Ti plasmid which is surrounded by the

20 two T-DNA borders is inserted by natural processes

into the plant genome. This segment of DNA contains
the chimeric gene and/or any other desired gene(s).
Preferably, the segment of vector DNA which is
inserted into the plant genome does not contain any

25 genes which would render the plant cell incapable of
being regenerated into a differentiated,
morphologically-normal plant. The transformed plant
cell(s) may be regenerated into a
morphologically-normal plant which will pass the

30 inserted gene to its descendants.

A variety of uses exist for plants
transformed by the method of this invention. For
example, a gene which codes for an enzyme which
35 inactivates a herbicide may be inserted into a plant.
This would make the plant and its descendants

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PCT/US84/00049

resistant to the herbicide. Alternately, a gene which
codes for a desired mammalian polypeptide such as
growth hormone, insulin, interferon, or somatostatin
may be inserted into plants. The plants may be grown
and harvested, and the polypeptide could be extracted
from the plant tissue.

BRIEF DESCRIPTION OF TEE DRAWINGS

FIG. 1 is a flow chart indicating the steps
of this invention, using pMON128 and the NOS-NFT II.
gene as an example.

FIG. 2 represents the creation of pM0N41, a
15 plasmid used to construct pMON120.

FIG. 3 represents the creation of M-4, an
M13-derived DNA used to construct pMON109.

20 FIG. 4 represents the creation of pMON54, a

plasmid used to construct pMON109.

FIG. 5 represents the creation of pMON109, a
plasmid used to construct pMON120.

FIG. 6 represents the creation of pMON113, a
plasmid used to construct pMON120.

FIG. 7 represents the creation of plasmid
30 pMON120, an intermediate vector with three restriction
endonuclease cleavage sites which are suitable for the
insertion of a desired gene.

FIG. 8 represents the creation of pMON128,
an intermediate vector which was obtained by inserting

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a chimeric NOS-NFT II kanamycin-resistance gene into
PMON120.

FIG. 9 represents the cointegration of
5 pMON128 with a wild-type Ti plasmid by means of a
single crossover event, thereby creating a
co-integrate plasmid with multiple borders.

FIG. 10 indicates the co-integration of
10 pMON128 with a disarmed Ti -plasmid, thereby creating a
non-tumorigenic cointegrate plasmid.

FIG. 11 is a graph comparing growth of
transformed cells and non- trans formed cells on
15 kanamycin-cont a i n i ng medium.

DETAILED DESCRIPTION OF THE INVENTION

In one preferred embodiment of this
invention, a variety of chimeric genes were inserted
into plant cells using the steps that are summarized
on the flow chart of Figure 1. As shown on Figure 1,
25 three preliminary plasmids were prepared. Those
plasmids were designated as:

1. pMON41, which contained a right border
from a nopaline-type Ti plasmid, and the 5 f portion -of

30 a nopaline synthase (NOS) gene. The construction of
plasmid pM0N41 is described below and shown in Figure
2. '

2. pMON109, which contained the 3 1 portion
35 of a NOS gene, and a selectable marker gene (spc/str)

which allowed for the selection of A. tumefaciens

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cells .having -co-integrate Ti plasmids with chimeric
genes- The construction of plasmid pMON109 is
described below and shown in Figures 3, 4, and 5.

3. pMON113, which contained a region of DNA
with a sequence that is identical to the sequence
within the T-DNA portion of an octopine-type Ti
plasmid. This region was designated as the "left
inside homology" (LIH) region- The construction of
pMON113 is described below and. shown in Figure 6.

After these plasmids were assembled, each
plasmid was digested by appropriate endonucleases to
obtain a desired fragment. Three fragments (one from
each of the three plasmids) were assembled in a triple
15 ligation to obtain the intermediate vector, plasmid
pMON120; as shown in Figure 7.

Plasmid pMON120 plays a key role in the
embodiment of this invention that is described in
detail below. This plasmid has the following
20 characteristics:

1. pMON120 has at least three unique
restriction endonuclease cleavage sites (EcoRI, Clal,
and Hindi II) which allow for the convenient insertion

25 of any desired gene.

2 . pMON120 will replicate within normal

E. coli - cells. However, it will not replicate within
normal Agrobacterium cells unless it co-integrates
30 with another plasmid, such as a Ti plasmid, which will
replicate in Agrobacterium cells.

3. pMON120 carries a marker gene which codes
for an enzyme which confers resistance to two

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antibiotics , spectinomycin (spc) and streptomycin
(stx). This gene, referred to as the spc/str gene, is
expressed in E. coli and in tumef aciens , but not in
plant cells. pMON120 does not carry genes which code
5 for resistance to ampicillin or tetracycline.

4. pMON120 carries a sequence which is
homologous to a sequence within the T-DNA portion of
an octopine-type Ti plasmid of A . tumef aciens > This

10 sequence is referred to as the "left inside homology"
(LIH) region* This region of homology promotes a
crossover event whereby pMON120, or a derivative of
pMON120 such as pMON128, forms a co-integrate with
the Ti plasmid if the two plasmids exist inside the

15 same A. tumef aciens cell. By definition, the

"co-integrate" plasmid is formed by a single crossover
event. It contains all DNA sequences that previously
existed in either the Ti plasmid or the
pMON120-derived plasmid.

5. pMON120 carries a nopaline-type T-DNA
"right border," i.e., a sequence which is capable of
acting as one end (designated by convention as the
right border) of a T-DNA sequence which is transferred

25 from a Ti plasmid and inseirted into the chromosome of
a plant cell during transformation of the cell by
A . tumef aciens. .

6. pMON120 carries a gene (including a

30 promoter) which codes for the expression of an enzyme,
nopaline synthase (NOS ) . Once introduced into a plant
cell, the NOS enzyme catalyzes the production of
nopaline, a type of opine. In most types of .plants,
opines are non-detrimental compounds which accumulate

35 at low levels; the presence of nopaline can be readily
detected in plant tissue (Otten and Schilperoort,

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1978). Opine genes may serve as useful marker genes to
confirm transformation, since 1 opines do not normally
exist in untrans formed plant cells. If desired, the
NOS gene in pMON120 may be rendered non- functional by
5 a variety of techniques known to those skilled in the
art. For example, a BamHI cleavage site exists within
the coding portion of the NOS gene; a stop codon or
other appropriate oligonucleotide sequence could be
inserted into this site to prevent the translation of
10 NOS .

7. , The relative location of the various
genes, cleavage sites, and other sequences in pMON120
is very important to the performance of this

15 invention. The entire pMON120 plasmid, or its

derivative plasmid such as pMON128, will be contained
in the co-integrate Ti plasmid. However, only part of
the co-integrate Ti plasmid {the modified T-DNA
region) will be inserted into the plant genome.

20 Therefore, only a part of the pMON120 -derived plasmid
will be inserted into the plant genome. This portion
begins at the T-DNA border, and stretches in one
direction only to the region of homology. In pMON120,
the NOS scorable marker, the spc/str selectable

25 marker, and the three insertion sites are within the
portion of pMON120 that would be transferred into the
plant genome. However, the pBR322-derived region next
to the LIH, and the Pvul cleavage site, probably would
not be transferred into the plant genome. Importantly,

30 this arrangement of pMON120 and its derivatives
prevents the transfer of more than one region of
homology into the plant genome, as discussed below.

8. pMON120 is about 8 kilobases long. This
is sufficiently small to allow it to accomplish all of
the objectives of this invention. H wever, if

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desired, it may be made somewhat smaller by the
deletion of one or more nucleotide sequences which are
not essential, using methods which are known to those
skilled in the art. Such a reduction in size might
improve the efficiency or other characteristics of the
plasmid when used for this invention or for other
purposes, as may be determined by those skilled in the
art. ■

It is recognized that a wide variety of
intermediate vectors which differ from pMON120 in one
or more respects may be prepared and utilized by those
skilled in the art. For example, the NOS marker used
for scoring transformed plant cells might be deleted,
or replaced or supplemented by a different scorable or
selectable marker. One such marker gene might
comprise an antibiotic- resistance gene such as the
NOS-NPT II-NOS chimeric gene described below. As
another example, the-spc/str marker gene used for
selecting A. tumefaciens cells with co-integrate
plasmids might be deleted, or replaced or supplemented
by a different scorable or selectable marker that is
expressed in Agxobacteria . As another example, a
variety of T-DNA borders (such as a nopaline-type
"left" border, or an octopine- type left or right
border) might be utilized. Similarly, more than one
border (such as two or more nopaline right borders, or
one nopaline right border and one octopine right
border) might be inserted into the intermediate
vector, in the desired orientation; this may increase
the frequency of insertion of the T-DNA into the plant
genome, as may be determined by those skilled in the
art. It is also possible to insert both left and
right borders (of any type) into an intermediate
vector. It is also possible to increase the length of
the region of hom logy; this is likely to increase the

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frequency of the desired single crossover event
(Leemans et al, 1981). It is also possible to select
an appropriate region of homology from any type of
desired plasmid, such as a nopaline or agropine Ti
plasmid or an Ri plasmid; such regions will allow the
intermediate vector to form a co-integrate with any
desired plasmid.

Method of Creating PMON120

Plasmid pMON120 was constructed from
fragments derived from 3 other plasmids. These three
plasmids were designated as pMON41, pMON109, and
pMON113. The construction of each of these three
15 plasmids is summarized below; additional information
is provided in the examples.

Plasmid PM0N41 contributed a nopaline-type
T-DNA right border and the 5' portion of a nopaline
20 synthase (NOS) gene to pMON120. It was created by the
following method.

A nopaline- type' Ti plasmid, designated as
the pTiT37 plasmid, may be digested with the Hindi 1 1

25 endonuclease to produce a variety of fragments,

including a 3.4 kb fragment which is designated as the
Hindi 1 1-23 fragment. This fragment contains the
entire NOS gene and the T-DNA right border. The
Applicants inserted a HindIII-23 fragment into a

30 plasmid, pBR327 (Soberon et al, 1980), which had been
digested with Hindlll. The resulting plasmid,
designated as pMON38, was digested with both Hindi I I
and BamHI. This produced a 2.3 kb fragment which
contains the nopaline-type right border and the 5 f

35 portion of a NOS gene ( including the promoter region,
the 5 1 non- translated region/ and part of the

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structural sequence ) - This 2.3 kb fragment was
inserted into a pBR327 plasmid which had been digested
with Hindu I and BamHI. The resulting plasmid was
designated as pM0N41, as shown in Figure 2.

A variety of strains of A. tumefaciens are
publicly available from the American Type Culture
Collection (Rockville, MD) ; accession numbers are
listed in any ATCC catalog- Each strain contains a Ti
10 plasmid which is likely to be suitable for use in this
invention, as may be determined through routine
experimentation by those skilled in the art.

Plasmid pMON109 contributed a spc/str
15 selectable marker gene and the 3 1 portion of a NOS
gene to pMON120. It was created by the following
method.

Plasmid pMON38 (described above and shown on
20 Figure 2) was digested with Rsal, which creates blunt
ends as shown: 5 ' -GTAC

CATC

A 1.1 kb fragment was isolated, and digested with
25 BamHI to obtain' fragments of 720 bp and 400 bp, each
of which had one blunt Rsa end and a cohesive BamHI
end. These fragments were added to double stranded
DNA from a phage M13 mp8 (Messing and Vieira, 1982)
which had been digested with Smal (which creates blunt
30 ends) and BamHI. The mixture was ligated, transformed
into cells and plated for recombinant phage.
Recombinant phage DNA's which contained the inserted
720 bp fragment were identified by the size of the
BamHI -Smal insert. One of those phage DNA's was
35 designated as M-4> as shown in Figure 3. The 720 bp
insert contained the 3 1 hon- translated region

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( including the poly-adenylation signal indicated in
the figures by a heavy dot) of the NOS gene, as well
as the 3' portion of the structural sequence of the
NOS gene. The 720 bp insert is surrounded in M-4 by
5 EcoRI and PstI cleavage sites, which were present in
the M13 mp8 DNA.

A bacterial transposon, designated as Tn7,
is known to contain the spc/str gene, mentioned

10 previously. The Tn7 transposon also contains a gene
which causes the host cell to be resistant to the
antibiotic trimethoprim. The exact location and
orientation of the spc/str gene and the
trimethoprim- resistance gene in Tn7, are not known.

15 The Tn7 transposon may be obtained from a variety of
cell strains which are publicly available. A strain
of A. tumefaciens was isolated in which the Tn7
transposon had been inserted into the Hind I I 1-23
region of a pTiT37 plasmid. The modified pTiT37

20 plasmid was designated as pGV3106 (Hernalsteens et al f
1980).

Plasmid pGV3106 was digested with Hindi II,
and the fragments were shotgun-cloned into pBR327

25 plasmids which had been digested with Hindlll. These
plasmids were inserted into E. coli cells, and cells
which were ampicillin-resistant (due to a pBR327 gene)
and trimethoprim-resistant (due to a Tn7 gene) were
selected. The plasmid obtained from one colony was

30 designated as pMON31. This plasmid contained a 6kb
Hindi I I insert. The insert contained the
spc/str-resistance gene and trimethoprim-resistance
gene from Tn7, and the 3' portion of a NOS gene (which
came from the pTiT37 plasmid).

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Plasmid pM0N31 was r due d in size twice.
The first reduction was performed by digesting the
plasmid with EcoRI, diluting the mixture to remove an
850 bp fragment/ and religating the large fragment.
5 The resulting plasmid, designated as pMDN53, was
obtained from transformed cells selected by their
resistance to ampicillin and streptomycin. Resistance
to trimethoprim was not determined.

10 Plasmid pMON53 was further reduced in size

by digesting the plasmid with Clal, diluting the
mixture to remove a 2 kb fragment, and religating the
large fragment. The resulting 5.2 kb plasmid was
designated as pMON54, as shown in Figure .4. This

15 plasmid contains the spc/str gene.

Plasmid pM0N54 was digested with EcoRI and
PstI, and a 4^8 kb fragment containing the spc/str
gene was isolated. M-4 DM was digested with EcoRI

20 and PstI, and a 740 bp fragment containing the NOS 3*
non-translated region was isolated. These fragments
were ligated together to form pMON64. In order to be
able to obtain the NOS 3 1 portion and the spc/str gene
on a single EcoRI-BamHI fragment, the orientation of

25 the spc/str gene was reversed by digesting pM0N64 with
Clal and religating the mixture. Plasmids having the
desired orientation were identified by cleavage using
EcoRI and BamHI. These plasmids wiere designated as
PMON109, as shown in Figure 5.

Plasmid PMON113 contributed a region of
homology to pM0N12G which allows pMON120 to form a
co-integrate plasmid when present in A. tumefaciens
along with a Ti plasmid. The region of homology was
35 taken from an octopine-type Ti plasmid. In the Ti
plasmid, it is located near the left T-DNA border,

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within the T-DNA portion of the Ti plasmid. This
region of homology is designated as the "left inside
homology" (LIH) region.

5 a region of homology may be derived from any

type of plasmid capable of transforming plant cells,
such as any Ti plasmid or any Ri plasmid. An
intermediate vector can be designed which can form a
co-integrate plasmid with whatever type of plasmid the
10 region of homology was derived from.

In addition, it might not be necessary for
the region of homology to be located within the T-DNA.
For example, it may be possible for a region of

15 homology to be derived from a segment of a Ti plasmid
which contains a T-DNA border arid a sequences of bases
outside of the T-DNA region. Indeed, if the
intermediate vector contains two appropriate T-DNA
borders, it might be possible for the region of

20 homology to be located entirely outside of the T-DNA
region.

The Applicants obtained an E. coli culture
with a pBR-derived plasmid containing the Bam-8

25 fragment of an octopine-type Ti plasmid. The Bam-8
fragment, which is about 7.5 kb, contains the left
border and the LIH region of the Ti plasmid
(Willmitzer et al, 1982; DeGreve et al, 1981). The
Bam-8 fragment was inserted into the plasmid pBR327,

30 which had been digested with BamHI. The resulting

plasmid was designated as pMON90, as shown in Figure
6.

Plasmid pMON90 was digested with Bglll, and
a 2.6 kb fragment which contains the LIH region but
not the left border was purified. The 2.6 kb fragment

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was treated with Klenow polymerase to convert the
cohesive ends into blunt ends, and the fragment was
digested with Hindi 1 1 to obtain a 1.6 Kb fragment (the
desired fragment) and a 1 kb fragment. Both fragments
5 were mixed with a pBR322 plasmid which had been
digested with PvuII and Hindi 1 1.. The mixture was
ligated, and inserted into E. coli cells. The cells
were selected for ampicillin resistance, and scored
for the presence of a Smal site which exists on the
10 1.6 Kb fragment but not the 1 Kb fragment. A colony
having the desired plasmid was identified, and the
plasmid from this colony was designated as pMON113, as
indicated by Figure 6.

15 To assemble pMON120, three fragments had to .

be isolated. Plasmid pM0N41 was digested with Pvul
and BamI, and a 1.5 Kb fragment containing a
nopaline-type right border and the 5 f portion of a NOS
gene was isolated: Plasmid pM0N109 was digested with

20 BamHI and EcoRI, and a 3.4 Kb fragment containing a>
spc/str gene and the 3 f part of a NOS gene was
isolated. Plasmid pM0N113 was digested with Pvul and
EcoRI, and a 3.1 Kb fragment containing the LIH region
was isolated.

The three fragments were mixed together and
ligated to form pMON120, as shown on Figure 7. A
culture of E. coli containing pMON120 has been
deposited with the American Type Culture Collection.
30 This culture has been assigned accession number 39263.

It is recognized that a variety of different
methods could be used to create pMON120 , or any
similar intermediate vector. For example, instead of
35 the triple ligation, it would have been possible to

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assemble two of the desired fragments in a plasmid f
and insert the third fragment into the plasmid.

Method of Using pMON120

As mentioned previously, pMON120 has three
unique cleavage sites (EcoRI, Clal, and Hindi II) which
are suitable for the insertion of any desired gene.
These cleavage sites are located in the portion of
10. pMON120 that will be inserted into a plant genome, so
the inserted gene also will be inserted into the plant
genome.

A variety of chimeric genes which are
capable of expressing bacterial and mammalian

15 polypeptides in plant cells have been created by the
Applicants . These chimeric genes are described in
detail in a separate application entitled "Chimeric
Genes Suitable for Expression in Plant Cells,"
copending U.S. application serial number 458,414,

20 filed on January 17, 1983.

Those chimeric genes are suitable for use in this
invention. They may be inserted into pMON120 to
create a derivative plasmid, which may be utilized as
described below,

25 In one preferred embodiment of this

invention, a chimeric gene was created which comprises
the following DNA sequences:

1. a promoter region and a 5 f non- translated
30 region derived from a nopalirie synthase (NOS) gene;

2. a structural sequence derived from a
n omycin phosphotransferase II (NPT II) gene; and

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3. a 3 f non- translated region derived from a

NOS gene.

5 This chimeric NOS-NPT 1 1 -NOS gene was

isolated on a DNA fragment having EcoRI ends. This
fragment was inserted into the EcoRI cleavage site of
pMON120, and the resulting plasmids (having chimeric
gene inserts with opposite orientations) were

i0 designated as pMON128 and pMON129, as shown in Figure
8. Plasmid pMON129 has two copies of the chimeric
gene; this may be a useful feature in certain types of
work. Either plasmid may be utilized to transform
plant cells, in the following manner- A culture of

15 E.coli containing pM0N128 has been deposited with the
American Type Culture Collection . . This culture has
been assigned accession number 39264.

20 Plasmid pMON128 (or any other plasmid

derived by inserting a desired gene into pMON120) is
inserted into a' microorganism which contains an
octopine-type Ti plasmid (or other suitable plasmid).
Suitable microorganisms include A . tumef aciens and

25 A. rhizoqeries which carry Ti or Ri plasmids. Other
microorganisms which might also be useful for use in
this invention include other species of Aqr obacterium ,
as well as bacteria in the genus Rhizobia . The
suitability of these cells, or of any other cells

30 known at present or hereafter discovered or created,
for use in this invention may be determined through
routine experimentation by those skilled in the art.

The plasmid may be inserted into the
microorganism by any desired method, such as
transformation (i.e., contacting plasmids with cells

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that have been treated to increase their uptake of
DNA) or conjugation with cells that contain the
pMON128 or other plasmids..

5 The inserted plasmid (such as pMGN128) has a

region which is homologous to a sequence within the Ti
plasmid. This "LIE" region of homology allows a
single crossover event whereby pMON128 and an
octopine-type Ti plasmid combine with each other to

10 form a co-integrate plasmid- . See,, e.g., Stryer,
supra , at p. 752-754. Normally/ this will occur
within the A. tumefaciens cell after pMON128 has been
inserted into the cell. Alternately, the
co-integrate plasmid may be created in a different

15 type of cell or i n vitro. , and then inserted into an
A. tumefaciens or other type of cell which can
transfer the co-integrate plasmid into plant cells.

The inserted plasmid, such as pMON128,
20 combines with the Ti plasmid in the manner represented
by Figures 9 or 10, depending upon what type of Ti
plasmid is involved.

In Figure 9, item 2 represents the T-DNA
portion of an octopine-type Ti plasmid. Item 4 t
represents the inserted plasmid, such as pM0N128. When
these two plasmids co-exist in the same cell, a
crossover event can occur which results in the
creation of co-integrate plasmid 6.

Co-integrate plasmid 6 has one left border
8, and two right borders 10 and 12. The two right
borders are designated herein as the "proximal" right
border 10 (the right border closest to left border 8),
and the "distal" right border 12 (the right border
that is more distant from left border 8. Proximate

f OMPI

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right border 10 was carried by plasmid 4; the distal
right border, was contained on Ti plasmid 2 before
co-integration.

.5 A culture of A. tumefaciens GV3111

containing a co-integrate plasmid formed by pMON128
and wild- type Ti plasmid pTiB653 has been deposited
with the American Type Culture Center. This culture
has been assigned accession number 39266.

- 10

When co-integrate Ti plasmid 6, shown in
Figure 9, is inserted into a plant cell, either of two
regions of DNA may enter the plant genome, T-DNA
- region 14 or T-DNA region 16.
15 '•' ' r — • ••• ■

T-DNA region 14 is bounded by left border 8
and proximate right border 10. Region*14 contains the
chimeric gene and any other genes contained in plasmid
4, such as the spc/str selectable m&rker and the NOS
20 scorable marker. However, region 14 does not contain
any of the T-DNA genes which would cause crown gall
disease or otherwise disrupt the metabolism or
regenerative capacity of the plant cell.

25 T-DNA region 16 contains left border 8 and

both right borders 10 and 12. This segment of T- DNA
contains the chimeric gene and any other genes
contained in plasmid 4. However, T-DNA region 16 also
contains the T-DNA genes which are believed to cause

30 crown gall disease.

Either of the foregoing T-DNA segments,
Region 14 or Region 16, might be transferred to the
plant DNA. This is presumed to occur at a 50-50
35 probability for any given T-DNA transfer. This is

likely to lead to a mixture of transformed cells, some

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of which axe tumorous .and some or which are
non- tumorous. It is possible to isolate and cultivate
non- tumorous cells from the mixture, as described in
the examples.

An alternate approach has also been
developed which avoids the need for isolating tumorous
from non- tumorous cells. Several mutant strains of
A.tumefaciens have been isolated which are incapable
10 of causing crown gall disease. Such strains are

usually referred to as "disarmed" Ti plasmids. A Ti
or Ri plasmid may be disarmed by one or more of the
following types of mutations:

15 i . Removal or inactivation of one of the

border. regions. One such disarmed octopine plasmid,
which has a left border but not a right border, is
designated as pAL4421; this plasmid is contained in
A. tumefaciens strain LBA4421 (Ooms et al, 1982;

20 Garfinkel et al, 1981)-

2. Removal or inactivation of the one or
more of the "tumor morphology" genes, designated as
the tmr and tms genes. See, e.g., Leemans et al,

25 1982.

Various other types of disarmed Ti plasmids
may be prepared using methods known to those skilled
in the art. See Matzke and Chilton, 1981; Leemans et
30 al, 1981; Koekman et al, 1979.

Figure 10 represents an octopine-type Ti
plasmid with a T-DNA region 22 which undergoes
mutation to delete the tms and tmr genes and the right
35 border. This results in a disarmed Ti plasmid with
partial T-DNA region 24. When plasmid 26 (such as

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pMON128) is inserted into a cell that carries the
disarmed Ti plasmid 24, a crossover event occurs which
creates a co-integrate Ti plasmid with disarmed T-DNA
region 28. The LIH region of homology is repeated in
5 this Ti plasmid, but the disarmed Ti plasmid does not ,
contain any oncogenic genes. Alternately, if only the -
right border had been deleted from T-DNA region 22,
then the tms and tmr genes and the octopine synthase
(OCS) gene would be contained in the co- integrate
1G disarmed Ti plasmid; however, they would have been
located outside of the T-DNA borders.

The disarmed co-integrate Ti plasmid is used
15 to infect plant cells, and T-DNA region 28 enters the
plant genome, as shown by transformed DNA 30. Plant
cells transformed by disarmed T-DNA 28 have normal
phytohormone metabolism, and normal capability to be
regenerated into differentiated plants.

After pMON128 is inserted into A.
tumefaciens cells, the desired crossover event will
occur in a certain fraction of the cells. Cells which
contain co-integrate plasmids (whether virulent or

25 disarmed) may be easily selected from other cells in
which the crossover did not occur, in the following
manner. Plasmid pMON120 and its derivatives contain a
marker gene (spc/str), which is expressed in
A. tumefaciens . However, these plasmids do not

30 replicate in A. tumefaciens . Therefore, the spc/str

marker gene will not be replicated or stably inherited
by A. tumefaciens unless the inserted plasmid combines
with another plasmid that can replicate in
A . tumefaciens . The most probable such combination,

35 due to the region of homology, is the co-integrate,
formed with the Ti plasmid. A. tumefaciens cells

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which contain this co- integrate plasmid can readily be
identified and selected by growth of the cells on
medium containing either spc or str, or both.

5 The Ti plasmid 28, shown in- Figure 10,

contains two LIH regions.* It is possible that
co-integrate Ti plasmids will undergo a subsequent
crossover event, wherein the two LIH regions will
recombine. This event is undesirable, since it can

10 lead to a deletion of the DNA between the LIH regions,
including the chimeric gene. However, this is not
likely to lead to serious difficulties, for two
reasons • First, this event is likely to occur at a
relatively low probability, such as about 10-2.

15 Second, plasmid pMON120 and its derivatives have been
designed so that the selectable marker gene (spc/str)
is located in the region of DNA that would be deleted
by the crossover event. Therefore, the selective
conditions that "are used to Identify 'and culture

20 Aqrobacteria cells containing co-integrate plasmids

will also serve to kill the descendants of cells that
undergo a subsequent crossover event which eliminates
the chimeric gene from the Ti plasmid-
^ ■

25 Only one of the LIH regions in the

co-integrate Ti plasmid will be inserted into the
plant genome, as shown in Figure 10.. This important
feature results from the design of pMON120, and it
distinguishes this co-integrate plasmid from undesired

30 co-integrate plasmids formed by the prior art. The
LIH region which lies outside of the T-DNA borders
will not be inserted into the plant genome. This
leads to at least two important advantages. First,
the presence of two LIH regions inserted into the

35 plant genome could result in crossover events which
would lead to loss of the inserted genes' in the

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transformed plant cells and their progeny. Second, the
presence of two regions of DNA homology can
significantly complicate efforts to analyze the DNA
inserted into the plant genome (Matzke and Chilton,
5 1981)-

After A. tumefaciens cells which contain the
co-integrate Ti plasmids with the chimeric genes have
been identified and isolated, the co-integrate

10 plasmids (or portions thereof) must be inserted into

the plant cells* Eventually, methods may be developed
to perform this step directly. In the meantime, a
method has been developed which may be used
conveniently- and with good results . This method is

15 described in two separate copending U.S* patent

"~ applications , entitled "Transformation of Plant Cells
by Extended Bacterial Co- Cultivation" serial number
458,413, and "Rapid Culture of Plant Protoplasts, "
serial number 458,412, both of which were filed on

20 January 17, 1983. The method described in those

applications may be briefly summarized as follows.

The plant cells to be transformed are
contacted with enzymes which remove the cell walls.

25 This converts the plant cells into protoplasts, which
are viable cells surrounded by membranes. The enzymes
are removed, and the protoplasts begin to regenerate
cell wall material. At an appropriate time, the A.
tumefaciens cells (which contain the co- integrate Ti

30 plasmids with chimeric genes) are mixed with the plant
protoplasts. The cells are co-cultivated for a period
of time which allows the A. tumefaciens to infect the
plant cells. After an appropriate co-cultivation
period, the A^ tumefaciens cells are killed, and the

35 plant cells are propagated.

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Planf cells which have been transformed
(i.e., cells which have received DNA from the
co-integrate Ti plasmids) and their descendants may be
5 selected by a variety of methods, depending upon the
type of gene(s) that were inserted into the plant
genome. For example, certain genes may cause various
antibiotics to be inactivated; such genes include the
chimeric NOS-NFT II-NOS gene carried by pMON128. Such
10 genes may serve as selectable markers; a group of
cells may be cultured on medium containing the
antibiotic which is inactivated by the chimeric gene
product, and only those cells containing the
selectable marker gene will survive.

A variety of genes may serve as scorable
markers in plant cells. For example, pMON120 and its
derivative plasmids, such as pMON128, carry a nopaline
synthase (NOS) gene which is expressed in plant cells.

20 This gene codes for an enzyme which catalyzes the

formation of nopaline. Nopaline is a non-detrimental
compound which usually is accumulated at low .
quantities in most types of plants; it can be easily
detected by electrophoretic or chromatographic

25 methods .

If a plant is transformed by a gene which creates
a polypeptide that is difficult to detect, then the
presence of a selectable marker gene (such as the
30 NOS-NPT II-NOS chimeric gene) or a scorable marker

gene (such as the NOS gene) in the transforming vector
may assist in the identification and isolation of
transformed cells.

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Virtually any desired gene may be inserted
into pMON120 or other plasmids which are designed to
form co-integrates with Ti or similar plasmids. For
example, the Applicants have created a variety of
5 chimeric genes, which are discussed in the

previously-cited application, "Chimeric Genes Suitable
for Expression in Plant Cells."

The suitability of any gene for use in this
10 invention may be determined through routine

experimentation by those skilled in the art. Such
usage is not limited to chimeric genes; for example,
this invention may be used to insert multiple copies
of a natural gene into plant cells. .

This invention is suitable for use with a
wide variety of plants, as may be determined through
routine experimentation by those skilled in the art.
For example, this invention is likely to be useful to

20 transform cells from any type of plant which can be

infected by bacteria from the genus Agrobacterium . It
is believed that virtually all dicotyledonous plants,
and certain monocots, can be infected by one or more
strains of Agr ob ac ter ium . In addition, microorganisms

25 of the genus Rhizobia are likely to be useful for

carrying co-integrate plasmids of this invention, as
may be determined by those skilled in the art. Such
bacteria might be preferred for certain types of
transformations or plant types.

Certain types of plant cells can be cultured
in vitro and regenerated into differentiated plants
using techniques known to those skilled in the art.
Such plant types include potatoes, tomatoes, carrots,
35 alfalfa and sunflowers. Research in in vitro plant

culture techniques is progressing rapidly, and methods

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ar likely to be developed to permit the regeneration
of a much wider range of plants from cells cultured
in vitro . Cells from any such plant with regenerative
capacity are likely to be transformable by the
in vitro co-cultivation method discussed previously,
as may be determined through routine experimentation
by those skilled in the art. Such transformed plant
cells may be regenerated into differentiated plants
using the procedures described in the examples.

T **e in vitro co-cultivation method offers
certain advantages in the transformation of plants
which are susceptible to in vitro culturing and

15 regeneration. However, this invention is not limited
to in vitro cell culture methods. For example, a
variety of plant shoots and cuttings (including
soybeans, carrots, and sunflowers) have been
transformed by contact with A . tumef aciens cells

20 carrying the co-integrate plasmids of this invention.
It is also possible to regenerate virtually any type
of plant from a cutting or shoot. Therefore, it may
be possible to develop methods of transforming shoots
or cuttings using virulent or preferably disarmed

25 co-integrate plasmids of this invention or mixtures

thereof, and subsequently regenerating the transformed
shoots or cuttings into differentiated plants which
pass the inserted genes to their progeny.

30 As mentioned previously, it is not essential

to this invention that co -cultivation be utilized to
transfer the co-integrate plasmids of this invention
into plant cells. A variety of other methods are
being used to insert DNA into cells . Such methods

35 include encapsulation of DNA in liposomes, complexing
the DNA with chemicals such as polycationic substances

OMR

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or calcium phosphate, fusion of bacterial spheroplasts
with plant protoplasts, microinjection of DNA into a
cell, and induction of DNA. uptake by means of electric
current. Although such methods have hot been used to
5 . insert DNA into plant cells with satisfactory -
efficiency to date r they are being actively researched
and they may be useful for inserting foreign genes *
into plant cells, using the intermediate vectors and
co-integrate plasmids of this invention, or plasmids
10 derived therefrom*

This invention may be useful for a wide
variety of purposes. For example, certain bacterial
enzymes, such as 5-enol pyruvyl shikimate-3 -phosphoric

15 acid synthase (EPSP synthase) are inactivated by
certain herbicides; other enzymes, such as
glutathione- S -trans f erase (GST), inactivate certain
herbicides. It may be possible to insert chimeric
genes into plants which will cause expression of such

20 enzymes in the plants, thereby causing the plants to
become resistant to one or more herbicides. This
would allow for the herbicide, which would normally
kill the untrans formed plant, to be applied to a field
of transformed plants. The herbicide would serve as a

25 weed-killer, leaving the transformed plants undamaged.

Alternately, it may be possible to insert
chimeric genes into plants which will cause the plants
to create mammalian polypeptides, such as insulin,
30 interferon, growth hormone, etc. At an appropriate ?
time, the plants (or cultured plant tissue) would be
harvested. Using a variety of processes which are *
known to those skilled in the art, the desired protein
may be extracted from the harvested plant tissue.

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An alternate use of this invention is to
create plants with high content of desired substances ,
such as storage proteins or other proteins * For
example, a plant might contain one or more copies of a
5 gene which codes for a desirable protein- Additional
copies of this gene may be inserted into the plant by
means of this invention. Alternately, the structural
sequence of the gene might be inserted into a chimeric
gene under the control of a different promoter which
10 causes prolific transcription of the structural
sequence.

The methods of this invention may be used to
identify, isolate, and study DMA sequences to

15 determine whether they are capable of promoting or
otherwise regulating the expression of genes within
plant cells. For example, the DNA from any type of
cell can be fragmented, using partial endonuclease
digestion or other methods. The DNA fragments can be .

20 . inserted randomly into plasmids similar to pMON128.

These plasmids, instead of having a full chimeric gene
such as NOS-NPT II-NOS, will have a partial chimeric
gene, with a cleavage site for the insertion of DNA
fragments, rather than a NOS promoter or other

25 promoter. The plasmids with inserted DNA are then

inserted into A. tumef aciens , where they can recombine
with the Ti plasmids. Cells having co-integrate
plasmids are selected by means of the spc/str or other
marker gene. The co-integrate plasmids are then

30 inserted into the plant cells, by bacterial

co— cultivation or other means. The plant cells will
contain a selectable marker structural sequence such
as the NPT II structural sequence, but this structural
sequence will not be transcribed unless the inserted

35 DNA fragment serves as a promoter for the structural
sequence. The plant cells may be selected by growing

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them on medium containing kanamycin or other
appropriate antibiotics.

Using this method, it is possible to
5 evaluate the promoter regions of bacteria, yeast ,

fungus, algae, other microorganisms, and animal cells
to determine whether they function as gene promoters
in various types of plant cells. It is also possible
to evaluate promoters from one type of plant in other

10 types of plant cells. By using similar methods and

varying the cleavage site in the starting plasmid, it
is possible to evaluate the performance of any DNA
sequence as a 5' non-translated region, a 3*
non-translated region, or any other type of regulatory

15 sequence.

As used herein, "a piece of DNA" includes
plasmids, phages, DNA fragments, and polynucleotides,
whether natural or synthetic.

As used herein, a "chimeric piece of DNA" is
limited to a piece of DNA which contains at least two
portions (i.e., two nucleotide sequences) that were
derived from different and distinct pieces of DNA.

25 For example, a chimeric piece of DNA cannot be created
by merely deleting one or more portions of a naturally
existing plasmid. A chimeric piece of DNA may be
produced by a variety of methods, such as ligating two
fragments from different plasmids together, or by

30 synthesizing a polynucleotide wherein the sequence of
bases was determined by analysis of the base sequences
of two different plasmids.

As used herein, a chimeric piece of DNA is
35 limited to DNA which has been assembled, synthesized,
or otherwise produced as a result of man-made efforts,

OMPI

Ukv wipo

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and any piece of DNA which is replicated or otherwise
derived therefrom. "Man-made efforts" include
enzymatic, cellular, and other biological processes,
if such processes occur under conditions which are
5 caused, enhanced, or controlled by human effort or
intervention; this excluses plasmids, phages, and
polynucleotides which are created solely by natural
processes. As used herein, the term "derived from"
shall be construed broadly. Whenever used in a claim,
10 the term "chimeric 11 shall be a material limitation.

As used herein, "foreign" DNA includes any
DNA which is inserted into a pre-existing plant cell.
A "foreign gene" is a gene which is inserted into a
15 pre-existing plant cell.

As used herein, a "marker gene" is a gene
which confers a phenotypically identifiable trait upon
the hbsit cell which allows transformed host cells to
20 be distinguished from non- trans formed cells. This

includes screenable, scorable, and selectable markers.

As used herein, a "region of homology"
refers to a sequence of bases in one plasmid which has

25 sufficient correlation with a sequence of bases in a
different plasmid to cause recombination of the
plasmid to occur at a statistically determinable
frequency. Preferably, such recombination should
occur at a frequency which allows for the convenient

30 selection of cells having combined plasmids, e.g.,

greater than 1 per 10 6 cells. This term is described
more fully in a variety of publications, e.g., Leemans
et al, 1981.

35 Those skilled in the art will recognize, or

be able to ascertain using no more than routine

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experimentation, numerous equivalents to the specific
embodiments of the invention discussed herein* Such
equivalents are within the scope of this invention.

Example 1: Creation of Plasmid pM0N41

A culture of E. coli , carrying a

10 pBR325 plasmid. (Bolivar, 1978) with the HindIII-23
fragment of pTiT37 (Hernal s teens , et al, 1980)
inserted at the Hindi 1 1 site, was obtained from Drs.
M. Bevan and M.D. Chilton, Washington University, St.
Louis, MO. Ten micrograms (ug) of the plasmid from

15 this clone was digested with 10 units of Hindlll

{unless noted, all restriction endonucleases used in
these constructions were purchased from New England
Biolabs, Beverly, MA and used with buffers according
to the supplier's instructions) for 1 hour at 37°C.

20 The 3.4 kb Hindi 1 1 -23 fragment was purified by

adsorption on glass beads (Vogelstein and Gillespie,
1979) after separation from the other Hindlll
fragments by electrophoresis on a 0.8% agarose gel.
The purified 3.4 kb Hindlll fragment (1.0 ug) was

25 mixed with 1.0 ug of plasmid pBR327 DNA (Soberon, et
al, 1980) that had been digested with both Hindlll (2
units, 1 hour, 37°C) and calf alkaline phosphatase
(CAP; 0.2 units, 1 hour, 37°C), de-proteinized with
phenol, ethanol precipitated, and resuspended in 10 ul

30 of TE (10 mM Tris HC1, pH8, 1 mM EDTA). One unit of
T4 DNA ligase (prepared by the method of Murray et al,
1979) was added to the fragment mixture. One unit is
defined as the concentration sufficient to obtain
greater than 90% circularization of one microgram of

35 Hindlll linearized pBR327 plasmid in 5 minutes at

22°C. The mixed fragments were contained in a total

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volume of 15 ul of 25 mM Tris-HCl pH8, 10 mM MgCl 2 , 1
mM dithiothreitol, 200 uM spermidine HC1 and 0.75 mM
ATP (ligase buffer).

5 The mixture was incubated at 22°C for 3 hours

and then mixed with . E. coli C600 recA 56 cells that
were prepared for transformation by treatment with
CaCl 2 (Maniatis et al, 1982). Following a period for
expression of the ampicillin resistance determinant

10 carried by the pBR327 vector, cells were spread on LB
solid medium plates (Miller, 1972) containing
ampicillin at 200 ug/ml. After incubation at 37°C for
16 hours, several hundred clones were obtained.
Plasmid mini -preps ( Ish-Horowicz and Burke, 1981) were

15 performed on 24 of these colonies and aliquots of the
plasmid DNA 1 s obtained : ( 6 . 1~ ug ) were digested with
Hindi 1 1 to demonstrate the presence of the 3.4 kb.
Hindi I I fragment. One plasmid demonstrated the
expected structure and was designated pMON38. pMON38

20 DNA were prepared by Triton X-100 lysis and CsCl
gradient procedure (Davis et al, 1980).

Fifty ug of pMON38 DNA were digested with
Hindlll and BamHI (50 units each, 2 hours, 37°c) and
the 2.3 kb Hindi 1 1 -BamHI fragment was purified as

25 described above. The purified fragment (1 ug) was

mixed with 1 ug of the 2.9 kb Hindi 1 1 -BamHI fragment
of the pBR327 vector purified as described above.
Following ligation (T4 DNA ligase, 2 units) and
transformation of E. coli cells as described above,

30 fifty ampicillin-resistant colonies were obtained.

DNAs from twelve plasmid mini -preps were digested with
Hindu I and BamHI to ascertain the presence of the 2.3
kb fragment. One plasmid of the correct structure was
chosen and designated pM0N41, as shown in Figure 2. A

35 quantity of this DNA was prepared as described above.

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Example 2 : Creation of M13 Clone M-4

Thirty ug of plasmid pMON38 (described in
Example 1) were digested with Rsal (30 units, 2 hours,
5 37°C) and the 1100 bp Rsal fragment was purified after
separation by agarose gel electrophoresis using the
glass bead method described in the previous example.
The purified 1100 bp Rsal -Rsal fragment (1 ug) was
digested with 2 units of BamHI and the BamHI was

10 inactivated by heating. This DNA was mixed with 0.2
ug of phage M13mp8RF DNA which had been previously
digested with Smal and BamHI (2 units each, 1 hour,
37°C) and 0.2 units of calf alkaline phosphotase (CAP) .
Following ligation with 100 units of T4 DNA ligase,

15 transformation of E. coli JM101 cells as described in
„-the_preyipM^ mixed
with soft agar and plated under conditions that allow
the identification of recombinant phage (Massing and >
Vieira, 1982). Twelve recombinant phage producing

20 . cells were picked and RF plasmid mini -preps were

obtained as described in the previous example. The RF
DNAs were digested with BamHI and Smal to prove the
presence of the 720 bp Rsal-BamHI fragment. One of
the recombinant RF DNAs carrying the correct fragment

25 was designated M13 mp8 M-4. This procedure is

represented in Figure 3. M-4 RF DNA was prepared
using the procedures of Ish-Horowicz and Burke, 1981
and Colman et al, 1978.

30 Example 3; Construction of PMON109

Twenty ug of plasmid pGV3106 (Heroalsteens
et al 1980, prepared by the method of Currier and
Nester 1976) was digested with Hindlll (20 units, 2
35 hours, 37°C) and mixed with 2 ug of Hindi 1 1 -digested

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pBR327. Following ligation (T4 DNA ligase, 2 units)
and transformation of E-. coli cells as described
above, one colony resistant to trimethoprim (100
ug/mi) and ampicillin was obtained. Digestion of
5 -plasmid DNA from this cell demonstrated the presence
of a 6 kb Hindi 1 1 fragment. This plasmid was
designated pMON31.

Plasmid pMON31 from a mini -prep (0.5 ug) was
10 digested with EcoRI (1 unit, 1 hour, 37°C) and the
endonuclease . was inactivated by heating (10 min,
70°C). The 8.5 kb plasmid fragment was
re-circularized in a ligation reaction of 100 ul (T4
DNA ligase, 1 unit) and used to transform E^ coli
15 cells with selection for ampicillin and streptomycin

(25 ug/ml) resistant colonies. Plasmid mini -prep DNA 1 s
from six clones were digested with EcoRI to ascertain
loss of the 850 bp fragment. One plasmid lacking the
. 850 bp EcoRI fragment was designated pMON53. This
20 plasmid was introduced into E. coli GM42 dam- cells
(Bale et al, 1979) by transformation as described.

Plasmid pMON53 (0.5 ug) from a mini -prep
prepared from dam- cells was digested with Clal, and

25 recircularized in dilute solution as described above.
Following transformation of E. coli GM42 cells and
selection for ampicillin and spectinomycin (50 ug/ml)
resistant clones, fifty colonies were obtained.
Digestion of plasmid mini -prep DNA T s from six colonies

30 showed that all lacked the 2 kb Clal fragment. One of
these plasmids was designated pMON54, as represented
in Figure 4. Plasmid DNA was prepared as described in
Example 1.

35 Plasmid pMON54 DNA (20 ug) was digested with

EcoRI and PstI (20 units of each, 2 hours, 37°C) and

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the 4.8 kb fragment was purified from agarose gels
using NA-45 membrane (Schleicher and Schuell, Keene,
N.H. ) .

5 The purified 4.8 kb fragment (0.5 ug) was

mixed with 0.3 ug of a 740 bp EcoRI -PstI fragment
obtained from M13mp8 M-4 RF DNA (described in Example
2) which was purified using NA-45 membrane. Following
ligation (T4 DNA ligase, 2 units), transformation of

10 E. coli GM42 dam- cells, and selection for

spectinomycin resistant cells, twenty colonies were
obtained. Plasmid mini -prep DNA 1 s prepared from
twelve of these clones were digested with PstI and
EcoRI to demonstrate the presence of the 740 bp

15 fragment. One plasmid carrying this fragment was

designated L pMON64. A guanti^ oif this plasmid DNA was
prepared as described in Example 1.

DNA (0.5 ug) of pMON64 was digested with
20 Clal (1 unit, 1 hour, 37°C), the, Clal was heat

inactivated, and the fragments rejoined with T4 DNA
ligase ( ^1 unit).. Following transformation and
selection for spectinomycin resistant cells, plasmid
mini-preps from twelve colonies were made. * The DNA's
25 were digested with BamHI and EcoRI to determine the
orientation of the 2 kb Clal fragment. Half of the
clones contained the Clal fragment in the inverse
orientation of that in pM0N64. One of these plasmids
was designated pMON109, as represented in Figure 5.
30 DNA was prepared as described in Example 1.

Example 4: Creation of plasmid PMON113

Plasmid pNW31C-8,29C (Thomashow et al, 1980)
35 was obtained from Dr. S. Gelvin of Purdue University,
West Lafayette, IN. This plasmid carries the pTiA6

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7.5 kb Bam- 8 fragment. The Bam-8 fragment was
purified from 50 ug of BamHI-digested pNW31C-8,2?C
using NA-45 membrane as described in previous
examples. The purified 7.5 kb Bam-8 fragment (1.0 ug)
5 was mixed with 0 .5 ug of pBR327 vector DNA which had
been previously digested with both BamHI (2 units) and
0.2 units of calf alkaline phosphatase (CAP) for 1
hour at 37°C; the mixture was deproteinized and
resuspended as described in previous examples. The

10 mixed fragments were treated with T4 ligase (2 units),
used to transform E. coli C600 recA cells and
ampicillin-resistant colonies were selected as
described previously. Mini-preps to obtain plasmid
DNA were performed on twelve of these clones. The DNA

15 was digested with BamHI to demonstrate the presence of
the pBR327 vector and 7.5 kb Bam-8 fragments. One
plasmid demonstrating both fragments was designated
pMON90. DNA was prepared as described in Example 1.

20 ' Twenty-five ug of pMON90 DNA were digested

with Bglll (25 units, 2 hours,^ 37°C) the 2.6 kb
Bglll fragment was purified using NA-45 membrane. To
create blunt ends, the fragment (2 ug) was resuspended
in 10 ul of 50 mM NaCl, 6.6 mM Tris-HCl pH8, 6.6 mM

25 MgCl 2 and 0.5 mM dithiothreitol (Klenow Buffer). The
4 deoxy-nucleoside triphosphates (dATP, dTTP ,~ dCTF,
and dGTP) were added to a final concentration of 1 mM.
and one unit of E. coli large Klenow fragment of DNA
polymerase I (New England Biolabs, Beverly, MA) was

30 added. After incubation for 20 minutes at 22°C, the

Klenow polymerase was heat inactivated and 10 units of
Hindi I I was added. The Hindi I I digestion was carried
out for 1 hour at 37°C and then the enzyme was heat
inactivated. The Hindlll-Bglll (blunt) fragments (1

35 ug) were added to 0.25 ug of the 2.2 kb Hindlll-PvuII
fragment of pBR322 (Bolivar, et al, 1977) which had

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been generated by Hindi 1 1 and PvuII digestion then
treating with calf alkaline phosphatase as described
in previous examples. After ligation using 100 units
of T4 DNA ligase, transformation of E. coli LE392
5 cells and selection of ampicillin-resistant colonies
as described in previous examples, nineteen colonies
were obtained. Plasmid mini -preps were prepared from
twelve colonies and digested with Hindi 1 1 to determine
the size of the recombinant plasmid and with Smal to
10 determine that the correct fragment had been inserted.
One plasmid with the correct structure was designated
pMON113 , as shown in Figure 6 . Plasmid DNA was
prepared as described in Example 1 *

15 Example 5: Creation of Plasmid PMON120

Twenty ug of plasmid pMON109 (described in
Example 3) were digested with EcoRI and BamHI (20
units each, 2 hours, 37°C) and the 3.4 kb BamHI -EcoRI

20 fragment was purified using NA-45 membrane as

described in previous examples. Twenty ug of plasmid
pM0N41 (described in Example 1) were digested with
BamHI and Pvul (20 units each, 2 hours, 37°C) and the
1.5 kb BamHI -Pvul fragment purified using NA-45

25 , membrane as described in previous examples.

Twenty ug of pMON113 DNA (described in
Example 4) were digested with Pvul and EcoRI (2 units
each, 2 hr, 37°C) and the 3.1 kb PvuI-EcoRI fragment

30 was purified using NA-45 membrane as above. To
assemble plasmid pMON120, the 3.1 kb EcoRI-PvuI
pM0N113 fragment (1.5 ug) was mixed with 1.5 ug of the
3.4 kb EcoRI -BamHI fragment from pMON109. After
treatment with T4 ligase (3 units) for 16 hours at

35 10°C, the ligase was inactivated by heating (10
minutes, 70°C), and 5 units of BamHI was added.

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Dig stion continued for 30 minutes at 37°C at which
time the BamHI endonuclease was inactivated by heating
as above. Next, 0.75 ug of the 1.5 kb PvuI-BamHI
fragment from pM0N4l was added along with T4 DNA
5 ligase ( 2 units) and fresh ATP to 0.75 mM final

concentration. The final ligase reaction was carried
out for 4 hours at 22°C at which time the mixture was
used to transform E. coli LE 392 cells with subsequent
selection for spectinomycin resistant cells as

10^ described previously* Plasmid mini-preps from twelve
out of several thousand colonies were screened for
plasmids of approximately 8 kb in size containing
single sites for BamHI and EcoRI. One plasmid showing
the correct structure was designated pMON120, which is

15 shown in Figure 7 with an alternate method of

construction. pMON120 DNA was prepared as described
in Example 1.

A culture of E. coli containing pMON120 has
20 been deposited with the American Type Culture

Collection. This culture has been assigned accession
number 39263.

Example 6; Creation of Plasmids pMON128 and
25 PMON129

Plasmid pMON75 (described in detail in a
separate application entitled "Chimeric Genes Suitable
for Expression in Plant Cells," previously cited)
30 contains a chimeric NOS-NPT II-NOS gene. This plasmid
(and pMON128, described below) may be digested by
EcoRI and a 1.5 kb fragment may be purified which
contains the NOS-NPT II-NOS gene.

35 Plasmid pMON120 was digested with EcoRI and

treated with calf alkaline phosphatase. After phenol

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deproteinization and ethanol precipitation, the
EcoRI-cleaved pMON120 linear DNA was mixed with 0.5 ug
of the 1.5 fcb EcoRI chimeric gene fragment from pMON75
or 76. The mixture was treated with 2 units of T4 DNA
5 ligase for 1 hour at 22°C. After transformation of
E. coli cells and selection of colonies resistant to
spectinomycin (50 ug/ml ) , several thousand colonies
appeared. Six of these were picked, grown, and plasmid
mini -preps made. The plasmid DNA's were digested with

10 EcoRI to check for the 1.5 kb chimeric gene insert and
with BamHI to determine the orientation of the insert.
BamHI digestion showned that in pMON128 the chimeric
gene was transcribed in the same direction as the
intact nopaline synthase gene of pMON120. A culture

15 of E. coli containing pMON128 has been deposited with
the American Type Culture Collection. This culture
has been assigned accession number 39264. The
orientation of the insert in pMON129 was opposite that
in pMON128; the "appearance of an additional 1.5 kb

20 BamHI fragment in digests of pMON129 showed that

plasmid pM0N129 carried a tandem duplication of the
chimeric NOS-NET II-NOS gene, as shown in Figure 8.

Example 7: Creation of Co-integrate Plasmid
25 PMON128: :pTiB6S3TraC

Plasmid pMON128 (described in Example 6) was
transferred to a chloramphenicol resistant
Agrobacterium tumefaciens strain GV3111=C58C1 carrying

30 Ti plasmid pTiB6S3tra c (Leemans, et al, 1982) using a
tri-parental plate mating procedure, as follows. 0.2
ml of a culture of E. coli carrying pMON128 was mixed
with 0.2 ml of a culture of E. coli strain HB101
carrying a pRK2013 plasmid (Ditta, et al, 1980) and

35 0.2 ml of GV3111 cells. The mixture of cells was

cultured in Luria Broth (LB), spread on an LB plate,

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and incubated for 16 to 24 hours at 30°C to allow
plasmid transfer and generation of co-integrate
plasmids. The cells were resuspended in 3 ml of 10 mM
MgS0 4 and 0.2 ml aliquot was then spread on an LB
plate containing 25 ug/ml chloramphenicol and 100
ug/ml each of spectinomycin . and streptomycin. After
incubation for 48 hr.at 30°C approximately 10 colonies
were obtained. One colony was chosen and grown at
30°C in LB medium containing chloramphenicol,
spectinomycin, and streptomycin at the concentrations
given above.

A separate type of co-integrate plasmid for
use in control experiments was prepared by inserting
pMON120 into A. tumefaciens cells, and selecting for
cells with co-integrate plasmids using spectinomycin
and streptomycin, as described above. Like pMON120,
these plasmids do not contain the chimeric NOS-NPT
II-NOS gene.

Example 8; Solutions Used in Plant Cell Cultures

The following solutions were used by the
Applicants:

per liter

Enzyme mix:

MS9:

Cellulysin
Macerozyme
Ampicillin

CaCI-

MgS0^.7H~0
KI * *
CuS0 4 .5H 2 0
Mannltol^

MS salts (see below)
Sucrose

5 g
0.7 g
0.4 g
27.2 mg
101 mg
1.48 g
246 mg
0.16 mg
0.025 mg
110 g

4.3 g
30.0 g

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B5 vitamins (see below) 1 ml

Mannitol 90.0 g
Phytohormones:

Benzyladenizie (BA) 0.5 mg

5 2,4-D 1 mg

MS-ES MS salts 4.3 g

Sucrose 30 g

B5 Vitamins " 1 ml

Mannitol 30 g

10 Carbenicillin 10 mg

Phytohormones:

Indole acetic acid 0*1 mg

MSO: MS salts 4.3 g

Sucrose 30.0 g

15 B5 vitamins 1 ml

Feeder plate MS salts 4.3 g

medium: Sucrose 30.0 g

B5 vitamins 1 ml

Mannitol 30.0 g
20 Phytohormones: -

BA 0.5 mg

Ms2C MS salts 4.3 g

Sucrose 30 g
B5 vitamins 1 ml

25 Phytohormones:

chlorophenoxyacetic 2 mg
acid

MS104: MS salts 4-3 g

Sucrose 30.0 g

30 B5 vitamins 1 ml

Phytohormones :

BA 0.1 mg

NAA 1 mg

MS 11: MS salts 4.3 g

35 Sucrose 30.0 g

B5 vitamins 1 ml
Phytohormones:

Zeatin 1 mg

B5 Vitamin myo.- inositol 100 g

40 stock: thiamine HCl 10 g

nicotinic acid 1 g -

pyrodoxine HCl 1 g

Float rinse: MS salts 0.43 g

Sucrose 171.2 g

45 PVP-40 40.0 g

MS salts are purchased pre-mixed
as a dry powder from Gibco Laboratories, Grand Island,
N.Y.

50 Example 9: Preparation of Protoplasts

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Mitchell petunia plants were grown in growth
chambers with two or three banks of fluorescent lamps
and two banks of incandescent bulbs (about 5,000 lux).
5 The temperature was maintained at a constant 21°C and
the lights were on for 12 hours per day- Plants were
grown in a 50/50 mix of Vermiculite and Pro-mix BX
(Premier Brands Inc., Canada). Plants were watered
once a day with Hoagland's nutrient solution. Tissue

10 was taken from dark green plants with compact, bushy
growth- Leaves were sterilized in a solution of 10%
commercial bleach and a small amount of detergent or
Tween 20 for 20 minutes with occasional agitation.
Leaves were rinsed two or three times with sterile

15 distilled water, Thin strips (about 1 mm) were cut

from the leaves, perpendicular to the main rib. The'
strips were placed in the enzyme mix at a ratio of
about 1 g tissue to 10 ml enzymes. The dishes were
sealed with parafilm, and incubated in the dark or

20 under low, indirect light while gently agitating
continuously (e.g., 40 rpm on gyrotary shaker).
Enzymic incubations generally were run overnight,
about 16-20 hours.

25 The digestion mixture was sieved through a

68, 74, or 88 urn screen to remove large debris and
leaf material. The filtrate was spun at 70-100 g for
five minutes to pellet the protoplasts. The
supernatant was decanted and the pellet was gently

30 resuspended in float rinse solution. This suspension
was poured into babcock bottles. The bottles were .
filled to 2 or 3 cm above the base of the neck. 1 ml
of growth medium MS 9 was carefully layered on top of
the float rinse.

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The Babcock bottles were balanced and
centrifuged at 500 to 1000 rpm for 10 to 20 minutes.
The protoplasts formed a compact band in the neck at
the interface. The band was removed with a pipette, ^
5 taking care not to pick up any excess float rinse.

The protoplasts were diluted into MS9. At this point,
the protoplasts were washed with MS 9 or. diluted for
plating without washing.

10 Protoplasts were suspended in MS 9 medium at

5 x 10 per ml, and plated into T-75 flasks, at 6 ml
per flask. Flasks were incubated on a level surface
with dim, indirect light or in the dark at 26-28°C.
On the third day following the removal of the enzymes

15 from the leaf tissue, MSO (medium which does not

contain mannitol ) -was added to each flask, using an
amount equal to one-half the original volume. The
same amount of MSO is added again on day 4. This
reduces the "mannitol concentration to about 0.33 M

20 after the first dilution, and about 0.25 M after the
second dilution.

Example 10: Co-cultivation with Bacteria

On day 5 following protoplast isolation,
25 five to seven day old tobacco suspension cultures (TXD
cells) were diluted (if necessary) with MS2C medium to
the point where 1 ml would spread easily over the
surface of agar medium in a 100 x 15 mm petri plate
[this is a 10 to 15% suspension (w/v)] . The agar
30 medium was obtained by mixing 0.8% agar with MS-ES
medium, autoclaving the mixture, and cooling the
mixture until it solidifies in the plate. One ml of
the TXD suspension was spread over 25 ml of feeder
plate medium. An 8.5 cm disc of Whatman #1 filter
35 paper was laid over the TXD feeder cells and smoothed

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out. A 7 cm disc of the same paper was placed in the
center of the larger one.

Separately, aliquots of a culture of A^
5 tumefaciens cells (grown in yeast extract peptone

medium) were added to the flasks which contained the
plant cells* One set of aliquots contained cells with
the pMON128::Ti co- integrate plasmids having. chimeric.
NOS-NPT II-NOS genes. The other set of aliquots
10 contained cells with the pMON120: :Ti co-integrate

plasmids, which do not have chimeric NOS-NPT II-NOS
genes v

The bacteria were , added to. the. flasks to- a^-
15 density of 10 8 cells/ml. 0.5 ml of the cell mixture
was spread in a thin layer on the surface of the 7 cm
filter paper disc. The plates were wrapped in
parafilm or plastic bags and incubated under direct
fluorescent lighting, no more than five plates in a
20 stack.

Within seven days, colonies were
discernable. Within 14 days, the 7 cm discs, with
colonies adhering to them, were transferred to new MSO

25 agar medium (without feeder cells) containing 500

ug/ml carbenicillin, as well as 50 ug/ml of kanamycih
sulfate (Sigma, St. Louis, MO), within two weeks,
vigorously growing green colonies could be observed on
the plates which contained plant cells that had been

30 co-cultured with A^ tumefaciens strains containing the
pMON128 co-integrate NOS-NPT II plasmid. No
transformed colonies were detected on plates which
contained plant cells that had been co-cultured with
A. tumefaciens strains containing the pMON120

35 co-integrate plasmid. The kanamycin resistant

trans formants are capable of sustained growth in

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culture medium containing kanamycin. Southern
blotting experiments (as described in £. Southern,
J. Mol. Biol. 98 : 503 (1975)) confirmed that these
cells contain the chimeric NOS-NPT II gene.

Both, sets of transformed cells (and a third
set of cells which had been transformed in the same
manner by a chimeric gene coding for the enzyme NPT
type I) were assayed for resistance to kanamycin. The
10 ... results are indicated in Figure 11.

Example 11: Regeneration of Transformed Plants

The transformed kanamycin^ resistant colonies
15 described in Example 10 contained both tumorous and
non- tumorous cells, as -.described in Figure 9 and the
related text. The following procedure was used to
isolate non- tumorous transformed cells from tumorous
transformed cells, and to regenerate differentiated
20 , plant tissue from the non-tumorous cells.

Colonies were grown on MS10.4 agar medium
containing 30 ug/ml kanamycin sulfate and 500 ug/ml
carbenicillin until they reached about 1 cm in
diameter. Predominantly tumorous colonies appear a

25 somewhat paler shade of green and are more loosely
organized than predominantly non-tumorous colonies
were removed from the MS 104 medium by tweezers and
placed upon MS11 medium containing 30 ug/ml kanamycin
and 500 ug/ml carbenicillin. As the colonies

30 continued to grow, colonies that appeared pale green
and loosely organized were removed and discarded.

MS 11 medium contains zeatin, a phytohormone
which induces shooting formation in non-tumorous
colonies. Several shoots were eventually observed

WO 84/02919

PCT/US84/00049

-53-

sprouting front kanaraycin-resistant colonies. These
shoots may be grown to a desired size, and cut off by
a sharp blade, and inserted into agar medium without
phytohormones , such as MSb, where it may generate
5 roots. If desired, the medium may be supplemented by
napthalene acetic acid to induce rooting. The plants
may be grown to a desired size in the agar medium r and
then transferred into soil. If properly cultivated,
such plants will grow to maturity and generate seed.
10 The acquired trait will be inherited by progeny
according to classic Mendelian genetics.

References ;

15 A. Bale et al, Mut. Res. 59 : 157 (1979)

F. Bolivar, Gene 4 : 121 (1978)

A. Braun and H. Wood, Proc. Natl. Acad. Sci. USA 73 :
496 (1976)

M. D. Chilton et al , Cell 11 : 263 (1977)
20 A. Colman et al, Eur. J. Biochem 91 : 303-310 (1978)

T. Currier and E. Nester, J. Bact. 126 : 157 (1976)
M. Davey et al, Plant Sci. Lett. 18 : 307 (1980)
R. w. Davis et al, Advanced Bacterial Genetics
Cold Spring Harbor Laboratory, New York, (1980)
25 H. De Greve et al, Flasmid 6 : 235 (1981)

G. Ditta et al, Proc. Natl. Acad. Sci. USA 77 : 7347
(1980)

D. Garfinkel et al, Cell 27 : 143 (1981)
S. Hasezawa et al, Mol. Gen. Genet. 182 : 206 (1981)
30 J . Hernalsteens et al, Nature 287 : 654 (1980),

D. Ish-Horowicz and J. F. Burke, Nucleic Acids
Res. 9 : 2989-2998 (1981)

B. Koekman et al, J.. 'Bacterid. 141 : 129 (1979)
F. Krens et al. Nature 296 : 72 (1982)

35 J. Leemans et al, J. Mol. Appl. Genet. 1 : 149 (1981)

WO 84/02919

PCT/US84/00049

J. Leemans et al, . The EMBO J. 1 ; 147 (1982)

A. L. Lehrunger, Biochemistry , 2nd ed. (Worth Publ..,
1975)

P. Lurquin, Nucleic Acids . Res. 6 ; 3773 (1979)
5 T. Maniatis et al r Molecular Cloning, A Laboratory

Manual (Cold Spring Harbor Labs, 1982)
L. Marton et al, Nature 277 : 129 (1979)
T. Matzke and M-D Chilton, J, Mol. Appl. Genet. 1 :
39 (1981)

10 J. Messing et al, Nucleic Acids Res. 9 ; 309 (1981)

J. Messing and J. Vieira, Gene 19: 269-276 (1982)
J. Miller, Experiments in Molecular Genetics , Cold

Spring Harbor Laboratory, N.Y. (1972)
N. Murray et al, J. Mol. Biol. 132 : 493 (1979)
15 G. Ooms et al, Plasmid 7 : 15 (1982)

L. Otten and R. Schilperoort, Biochim. Biophys

Acta 527 ; 497 (1978)
L. Otten, Mol. Gen, Genet. 183 : 209 (1981)
R. Roberts, Nucleic Acids Res. 10 : r!17 (1982)
20 A. Rorscfa and R. Schilperoort, Genetic Engineering ,

189 Elsevier/North Holland, N.Y. (1978)
J. K. Setlow and A. Hollaender, Genetic

Engineering, Principles and Methods (Plenum Press

1979)

25 X. Soberon et al, Gene 9 :' 287 (1980)

L. Stryer, Biochemistry , 2nd. ed. (W. H. Freeman and
Co., 1981)

M. Thomashow et al, Cell 19 : 729 (1980)

B. Vogelstein and D. Gillespie, Proc. Natl. Acad.
30 Sci. i 615-619 (1979)

L. Willmitzer et al. Nature 287 : 359 (1980)
L. Willmitzer et al. The EMBO J 1 : 139 (1982) r

WO 84/02919

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N. Yadev et al, Nature 287 : 1 (1980)
F.. Yang et al, Mol. Gen. Genet. 177 : 707 (1980)
P. Zambryski et al, J. Mol. Appl. Genet. 1 : 361
(1982)

WO 84/02919

PCT7US84/00G49

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CLAIMS

1. A chimeric plasmid comprising the
following elements, in sequence:

5 a. a first sequence of bases which is

replicated or derived from a first T-DNA border;
b. a second sequence of bases comprising one or
more genes which are expressed in one or more
types of plant cells ;
10 c. a third sequence of bases which

comprises a region of homology with a Ti plasmid,
wherein the third sequence is homologous to a
selected sequence of bases which is located in a
wide-type Ti plasmid between:
15 (1) a T-DNA border which is complementary

to. the first T-DNA border, and
(2) one or more tumori genie genes on the
Ti plasmid.

2. A chimeric plasmid of Claim 1,

20 comprising a gene which functions as a marker in one
or more types of microorganisms in which the Ti
plasmid replicates.

3. A chimeric plasmid of Claim 2,

wherein the gene functions as a selectable marker.
25 4. A chimeric plasmid of Claim 1,

comprising a gene which functions as a marker in
one or more types of plant cells*

WO 84/02919

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-57-

5. A chimeric plasmid of Claim 4,
wherein the gene functions as a scorable marker*

6. A chimeric plasmid of Claim 5

wherein at least one gene is expressed in plant cells
5 into a polypeptide which catalyzes the production of
one or more opine substances in plant cells.

7. A chimeric plasmid of Claim 4

wherein the gene functions as a selectable marker.

a. A chimeric plasmid of Claim 1

10 comprising a first gene which functions as a marker
in one or more types of microorganisms which are
capable of containing the Ti plasmid and a second
gene which functions as a marker in one or more types
of plant cells.

15 9. A chimeric plasmid of Claim 1, which

does not replicate in one or more types of
microorgansims in which the Ti plasmid replicates.

10. A chimeric plasmid comprising the
following elements , in sequence: ,

20 a. a first sequence of bases which is

replicated or derived from a first T-DNA border;

b. a second sequence of bases comprising one or
more genes which are expressed in one or more
types of plant cells; and

25 c. a third sequence of bases which comprises

a region of homology with a Ti plasmid,
wherein the third sequence is the only sequence of
bases havnig substantial homology with any sequence
of bases within the Ti plasmid.

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11. A chimeric plasmid of Claim 10.,
comprising a gene which functions as a marker in one
or more types of microorganisms in which the Ti
plasmid replicates .
5 12. A chimeric plasmid of Claim 11, ?

wherein the gene functions as a selectable marker.

13 . A chimeric plasmid of Claim 10,, *
comprising a gene which functions as a marker in
one or more types of plant cells.
10 14. A chimeric plasmid of Claim 13

wherein the gene functions as a scorable marker.

15 . A chimeric plasmid of Claim 13
wherein the gene is expressed in plant cells into
a polypeptide which catalyzes the production of
15 one or more opine substances in plant cells.

16. A chimeric plasmid. of Claim 13

wherein the gene functions as a selectable marker.

17. A chimeric plasmid of Claim 10
comprising a first gene which functions as a

20 marker in one or more types of microorganisms in
-> which the Ti plasmid replicates and a second gene
which functions as a marker in one or more types
of plant cells.

18. A chimeric plasmid, comprising the
25 following elements , in sequence:

a. a first sequence of bases which is
capable of serving as a first T-DNA border;

b. a second sequence of bases comprising
a gene which is expressed in plant cells;

30 c. a third sequence of bases which *

comprises a region of homology with a Ti plasmid,

WO 84/02919

PCT/US84/00049

-59-

wherein the chimeric plasmid is capable of forming
a co-integrate plasmid with the Ti plasmid by
means of a single crossover event at the region of
homology, wherein the co-integrate plasmid will
5 contain a fourth sequence of bases comprising the
first T-DNA border, the second seguence of bases,
and a complementary T-DNA border, wherein the
fourth seguence of bases will not contain any
tumorigenic genes.

10 19. A chimeric plasmid of Claim 18,

comprising a gene which functions as a marker in
one or more types of microorganisms in which
the Ti plasmid replicates.

20. A chimeric plasmid of Claim 18,

15 comprising a gene which functions as a marker in
one or more types of plant cells.

■21. A chimeric plasmid of Claim 18
comprising a first gene which functions as a
marker in one or more types of microorganisms

20 which the Ti plasmid replicates and a second
gene which functions as a marker in one or
more types of plant cells.

22. A chimeric plasmid comprising
the following elements:

25 a. a first seguence of bases which is

replicated or derived from a first T-DNA border;

b. a gene which is expressed- in plant
cells; and,

c. a second sequence of bases which is

30 replicated or derived from a second T-DNA border

which is complementary to the first T-DNA border,

WO 84/02919 PCT/US84/00049

-60-

vherein there are no base sequences between the
two T-DNA borders which would render a host plant
cell tumorous or incapable of regeneration into a
morphologically normal plant*
5 23. A plasmid containing the chimeric plasmid

of Claim 22.

24. A plasmid of Claim 23 which is obtained
by inserting DNA into a Ti plasmid.

25. A plasmid of Claim 24 which contains a
10 gene which functions as a marker in one or more

types of microorganisms in which the Ti plasmid
replicates.

26. A plasmid of Claim 24 which contains a

gene which functions as a marker in one or more types
15 of plant cells.

27. A plasmid of Claim 24 which contains a first
gene which functions as a marker in one or more types
of microorganisms in which the Ti pl&smid replicates
and a second gene which functions as a

20 marker in one or more types of plant cells.

28. A microorganism which contains a
chimeric plasmid of Claim 1.

29. A microorganism which contains a
chimeric plasmid of Claim 8.

25 30. A microorganism which contains a

chimeric plasmid of Claim 18.

31. A microorganism which contains a
chimeric plasmid of Claim 21.

32. A microorganism which contains a
30 chimeric plasmid of Claim 22.

33 - A microorganism which contains a
chimeric plasmid of Claim 27.

WO 84/02919

-61-

PCT/US84/00049

34. A plasmid having the relevant charac-
teristics of plasmids contained within a culture of
cells having an ATCC accession number selected from the
group consisting of 39263, 39264, 39266.
5^ 35. A culture of cells descended from a

culture, of cells having an ATCC accession number
selected from the group consisting of 39263, 39264,
39266.

WO 84/02919

PCT/US84/00049

l/l I

ISOLATE DNA SEQUENCE WITH MARKER GENE FOR
SELECTION OF A. TUM. CONTAINING COINTEGRATE
PLASM I OS

(pMON 109)

ISOLATE ONA SEQUENCE WITH
T-DNA BORDER FROM Tl PLAS-
MIO ( optional: PLANT
SCORABLE MARKER)

(pMON 41)

ISOLATE DNA SEQUENCE WITH
REGION HOMOLOGOUS TO PART
OF T-DNA OF Tl PLASMID

(pMON 113)

LIGATE 3 SEQUENCES IN PROPER ORIENTATION "|

(pMON 120)

| INSERT CHIMERIC GENE INTO PLASMID \

.(pMON 126)

I NSERT PLASMID INTO ItCOU.;
MATE E. C OLI ; WITH A .TUMEFACIENS

INSERT PLASMID INTO A. TUMEFACIENS
CONTAI NING (DISARMED) Tl PLASMID

SELECT A. TUMEFACIENS CELLS WITH
CO-INTEGRATED Tl PLASMIDS HAVING
CHIMERIC GENES

CO- CULTIVATE A. TUMEFACIENS WITH
PLANT CELLS; ALLOW TRANSFER OF
CO-INTEGRATE PLASMIDS INTO PLANT
CELLS

CULTIVATE PLANT (
CELLS HAVING(EXF
GENE OR SCORABl

SELL .IDENTIFY PLANT
SESSION 0F)CHIMER1C
£ MARKER

REGENERATE PLANT CELLS INTO PLANTS j

FIG. I.

SUBSTITUTE SHEET

WO 84/02919

PCT/US84/00049

2/11

LEFT
BORDER

Hind III
Bom HI

FIG. 2,

Hind III

DIGEST WITH Hind III
PURIFY 3.4Kb Hind 111-23
FRAGMENT

Bom HI

Pvu I Hind III

tr\ 1

Hind III

RIGHT
BORDER

NOS GENE

DIGEST WITH /

Hind III CMIX.LIGATE, TRANSFORM,

CAP \ SELECT AmpRCELLS

Pvu I

Hind III

Bam HI

*RIGHT
BORDER

Bom HI

DIGEST WITH / « Hind III

BamHI, Hind III /DIGEST WITH
PURIFY 2.9 Kb / Bam H I , Hind III
FRAGMENT | PURIFY 2.3 Kb
FRAGMENT •

MIX, LIGATE.TRANSFORM,
SELECT Amp R CELLS

Pvu I

RIGHT
BORDER

Bom HI

SUBSTITUTE SHEET

Utv -woo ,»y

WO 84/02919

PCT/US84/00049

3/1 1

Hind til

DIGEST WITH Rsa I
PURIFY NOObp
FRAGMENT

R so I _ B _ Rso I

l Clo Bam HI i

01 _ u _ NOPALINE SYNTHASE

»«o Jcro 3' PORTION

BORDER

Hind III

Rso l-^CIa I

Rsa I
Bam HI

Sma I
EcoRI- I ' Bom H l

DIGEST
Bam HI

Rsa I

FIG.3.

Clo I Bam HI BamHI

• — i t=i

NOS 3*
DIGEST Sma l t PORTION
BamHI CAP

MIX.LIGATE.IDENTIFY
RECOMBINANT PHAGE

EcoRI-i Cla I

NOS POLY- A
SIGNAL

SUBSTITUTE SHEET

WO 84/02919

PCT/US84/00049

4/11

pGV3l06 = pTiT37 nosi: Tn7

§UMTITUtC SHEET

WO 84/02919

PCT/US84/00049

5/11

EcoRI

Clal

Bom HI

DIGEST WITH Clof
LIGATE.TRANS -
FORM, SELECT
SpcR CELLS

EcoRI

Clo I

FIG. 5.

SUBSTITUTE SHEET

OMR

K> wipo

WO 84/02919

PCT/US84/00049

6/1!

OCTOPINE-TYPE Ti PLASMID T-DNA

Bom -8

.T-DNA
* .RIGHT
/BORDER

Bam I

> T-DNA BamH! ' Bam HI

LI ' '

LEFT BORDER

BamHI

Bam H t

p Ti A6 FRAGMENT CLONED IN
p BR 322 ATTHE BamHI SITE
TO YIELD pNW 3IC -8, 29-1

^DIGEST pNW 310-8,29-1 DNA
.WITH Bam HI PURIFY THE 7.5Kb
BamHI FRAGMENT

DIGEST WITH
BamHI , CAP

w MIX,LIGATE,TRANSFORM f
SELECT Amp* CELLS

BamHI

Bgl II

DIGEST WITH Bgl II
Bgl II PURIFY 2.6Kb
Sma. Hind III ^ FRAGMENT
I

■ i

CREATE BLUNT ENDS
WITH KLENOW POLY-
MERASE + 4dNTPs Bflm

DIGEST WITH Hind III

Bgl II

Hind III

Bgl II

Bgl II (BLUNT)

Bgl 11/ Pvu II

Sma I

L DIGEST Pvu II, Hind III
PURIFY 2.2 Kb
i FRAGMENT

MIX, LI GATE,
TRANSFORM.
SELECT Amp*
CELLS

Sm0 1 Hind III
Pvu II

Hind III — M 1 — EcoRI
Clal

FIG. 6.

Hind III - J | , -EcoRI
Clol

SUBSTITUTE SHEET

WO 84/02919

PCT/US84/00049

7/1 1

Bam HI

EcoRI

DIGEST WITH Bam H I, EcoRI
PURIFY 3.4Kb FRAGMENT

Pvu I

Bam HI

EcoRI

DIGEST WITH Pvu l f
Bam HI
PURIFY 1.5Kb
FRAGMENT

Bam HI BamHI Pvu I

- — I I » i

spe/s,rR JJoT*

NOS 5'
> PORTION PORTION

Pvu I

EcoRI

DIGEST
Pvu I, EcoRI
PURIFY 3.1 Kb \
FRAGMENT

pTiT37
RIGHT BORDER

Pvu I

r-> tr * ~* X NOPALINE

rKj.f. / SYNTHASE

MIX, LIGATE,TRANSFORM f
SELECT Spc* CELLS -

BamHI

Hind III ^I^EcoRI
Cla I

SUBSTITUTE SHEET

WO 84/02919

PCT/US84/00049

8/11

EcoRI

TRANSFORM CELLS
SELECT Spc* CELLS

NOS-NPTII- NOS NOS NOS

SUBSTITUTE SHEET

WO 84/02919

PCT/US84/00049

9/11

SINGLE- CROSSOVER-

COINTEGRATION

CHIMERIC Spc/
GENE n 'Str "

NOS

LIHl

tms
tmr

INSERTION OF T-DNA
INTO PLANT GENOME

- nos'I hIv^aaaa

NON-TUMORIGENIC T-DNA
OR

TUMORIGENIC T-DNA

F/G.9.

SUBSTITUTE SHEET

OMRI

WO 84/02919

PCT/US84/00049

10/11

tms
trnr

OCS

DELETION OF TUMORIGENIC
GENE(S) AND/OR T-DNA
RIGHT BORDER

SINGLE -CROSSOVER
CO I NTEG RATI ON

CHIMERIC
GENE

INSERTION OF T-DNA
INTO PLANT GENOME

vwvaO ^IThT - CH P ^g' c - s % r -|nos>»vwvv

FIG. 10.

SUBSTITUTE SHEET

WO 84/02919

11/11

PCT/US84/00049

100 "" 200

KANAMYGIN (ug/ml)

FIG J I

SUBSTITUTE SHEET

INTERNATIONAL SEARCH REPORT .

International Application No PCT/US84/0004 9

I. CLASSIFIC ATION OP SUBJECT MATTER (If aeveral classification symbols apply, Indicate all) »
According to Iffornatlonal Patent Classification (IPC) or to both National Classification and. IPC

Int. C1?C12N 15/00, 1/20, 1/00 <

II. FIELDS SEARCHED

Minimum Documentation Searched *

Classification System

Classification Symbols

U.S.

435/172, 253, 317

Documentation Searched other than Minimum Documentation
to the Extent that such Documents am included In the Fields Searched >

DOCUMENTS CONSIDERED TO BE RELEVANT **

Cateoory * I Citation of Document *» with indication, where appropriate, of the relevant passages *

N, Schell et al., Genetic Engineering to
Biotechnology - The Critical Transition,
Edited by W.J. Whelan and Sandra Black,
Published by John Wiley & Sons Ltd.,
pages 41-52 (19&2)

N, Zambryski et al. , Journal of Molecular
and Applied Genetics, Vol. 1, No. 4
pages 361-370 (1982)

N, Matzke et al., Journal of Molecular and
Applied Genetics, Vol. 1, No. 1, pages
39-49* (1981)

N, Leemans et al. , Journal of Molecular and
Applied Genetics, Vol. 1, No. 2, pages
149-164 C1981)

N, Leemans et al. , Molecular Biology of
Plant Tumors, edited by G. Kahl and
J.S. Schell, published by Academic Press
Inc., pages 537-545 (1982)

1-35

* Special categories of cited documents: "

•A" document defining the general state of the art which Is not

considered to be of particular relevance
"E" eariler document but published on or after the International

flflng date

"L" document which may throw doubts on priority ^aimjs) or
which Is cited to establish the publication date of another
citation or other special reason (as specified)

-cr document referring to an oral disclosure, us* exhibition or
other means

M P" document published prior to the international filing date but
later than the priority date claimed

-T- later document published after the Interzonal flHng date
or oriority date and not in conflict with the ^Pjteg 0 " JJJ
cited I to undersSnd the principle or theory underlying the
Invention

»x» document of particular relevance; the claimed InwnMon
^ caJJS be considered novel or cannot be considered to
involve an Inventive step

«Y- document of particular relevance; *»j£ m «l £22 tt |E
ST* considered to Involve an inventive step when tne

In th» art

"A* document member of the same patent family

IV. CERTIFICATION

Date of the Actual Completion of the International Search '

4 April 1984

Date of Mailing of this International Saarcn Report »

16 APR 1984

International Searching Authority 3

ISA/US

Slgntfurs^f AuthortoedJ2(ftcer < «

AlvinETTa^iehho It z

Form PCT/ISA/210 (second sheet) (October 1981)

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